Tissue specificity and evolution of meristematic WOX3 function

Abstract

The WUSCHEL-related homeobox (WOX) gene PRESSED FLOWER1 (PRS1) performs a conserved function during lateral organ development in Arabidopsis (Arabidopsis thaliana). Expressed in the periphery of the shoot meristem, PRS1 recruits founder cells that form lateral domains of vegetative and floral organs. Null mutations in PRS1 cause the deletion of lateral stipules from leaves and of lateral sepals and stamens from flowers. Although PRS1 expression is described in the L1 layer, PRS1 recruits founder cells from all meristem layers. The mechanism of non-cell autonomous PRS1 function and the evolution of disparate WOX gene functions are investigated herein. Meristem layer-specific promoters reveal that both L1 and L1-L2 expression of PRS1 fail to fully rescue PRS1 function, and PRS1 protein does not traffic laterally or transversely between shoot meristem layers. PRS1 protein accumulates within all meristematic cell layers (L1-L2-L3) when expressed from the native promoter, presumably due to low-level transcription in the L2 and L3 layers. When driven from the PRS1 promoter, full rescue of vegetative and floral prs1 mutant phenotypes is provided by WUSCHEL1 (WUS1), which is normally expressed in the stem cell organizing center of shoot meristems. The data reveal that WUS1 and PRS1 can engage in equivalent protein-protein interactions and direct transcription of conserved target genes, suggesting that their subfunctionalization has evolved primarily via diverse promoter specificity. Unexpectedly, these results also suggest that meristematic stem cells and lateral organ founder cells are intrinsically similar and formed via equivalent processes such that their ultimate fate is dependent upon stage-specific and domain-specific positional signaling.

Figure 1.

Expression of PRS1 in the L1 and L1-L2 histological layers fails to complement prs1 mutant phenotypes. A, Whole plant phenotypes of (left to right) prs1 non-mutant (Ler), prs1 mutant (prs), and transgenic plants expressing PRS∼GFP fusion proteins driven by the native PRS1 promoter (C1 PRS), the L1-specific promoter ML1 (C2 L1), and the L1-L2-layer-specific promoter SCR1 (C3 L1L2). Note that the short plant stature phenotype seen in prs mutant plants is complemented in C1 PRS plants; partial complementation is seen in C3 L1-L2 plants, and still weaker complementation is observed in C2 L1 plants. B to F, Stage 12 floral phenotypes (i.e. prior to bud opening; Smyth et al., 1991) of plants shown in A. Whereas Ler (B) and C1 PRS (D) flowers have four sepals of normal width, prs1 mutant (C) flowers typically fail to develop lateral sepals. E, C2 L1 flowers exhibit incomplete rescue of prs1 mutant floral phenotypes. Although C3 L1-L2 (F) flowers form four sepals, the lateral sepals are narrow and fail to fully enclose the underlying floral organs. G to K, Transverse sections of shoot apices of vegetative seedlings show development of lateral stipules (arrows) in rosette leaves of Ler (G) and C1 PRS (I) plants, but not in prs1 mutants (H), C2 L1 plants (J), or C3 L1-L2 plants (K). L to N, Lateral stipule (arrow) formation at the base of cauline leaves in Ler plants; prs1 mutant (M) and C3 L1-L2 plants (N) fail to form stipules on cauline leaves. CL, Cauline leaf; FB, floral bud; ad, adaxial sepal; ab, abaxial sepal; l, lateral sepal; st, stamen; gy, gynoecium; IS, inflorescence stem. Bars in A = 3 cm; B (for B to F) = 100 μm; G (for G to K) = 50 μm; L (for L to N) = 50 μm.

Figure 2.

Expression constructs used in this study. Promoter regions are depicted as black bars; open reading frames are shown as colored rectangles. Not to scale. Plasmid details are provided in “Materials and Methods.”

Figure 3.

Sepal initiation and morphology are disrupted by loss of PRS1 function. Total number of sepals initiated (A) and sepal size relative to Ler floral buds (B) are reduced in prs1 mutant flowers, but are restored to near normal levels in C1 PRS1 and C5 WUS1 transgenic mutant plants. Although sepal number is improved in C2 L1 and C3 L2L3 transgenics (A), the sepal size phenotype is not complemented (B).

Figure 4.

Layer-specific accumulation of PRS∼GFP fusion proteins. Confocal imaging of top view (A) of stage 3 floral meristem in C1 PRS transgenic plants reveals accumulation of PRS1∼GFP fusion protein (arrow) in two lateral foci and in multiple cell layers. B, Side image of lateral domain of C1 PRS floral meristem. C, When expressed from the ML1 promoter, PRS1∼GFP accumulation is observed only in the L1 layer of C2 L1 transgenic plants. D, PRS1∼GFP fusions expressed from the SCR1 promoter are observed in L1-L2 layers of the C3 L1-L2 floral meristem. l, Lateral domain; ab, abaxial domain; ad, adaxial domain. Bar = 25 μm.

Figure 5.

Functional analyses and accumulation of PRS1∼GUS fusion proteins expressed from the native PRS1 promoter. C4 GUS transgenic prs1 mutant plants exhibit normal plant height (A) and normal sepal development (D) compared to prs1 mutant (C) and Ler (B) flowers. E and F, GUS staining of C4 GUS inflorescences reveal accumulation of PRS1∼GUS fusion proteins in the margins of cauline leaves, in stage 1 floral meristems, and in stage 3 floral meristems. G to J, Serial sections from the crown (G) of a stage 3 floral meristem in C4 GUS transgenic plant into medial sections (J) reveal a stripe of PRS1∼GUS accumulation across the meristem dome that bypasses the meristem median region at two lateral foci. CL, Cauline leaf; IM, inflorescence meristem; ad, adaxial domain; ab, abaxial domain; l, lateral domain; 1, stage 1 floral meristem; 2, stage 2 floral meristem; 3, stage 3 floral meristem. Bars in A = 3 cm; B (for B to D) = 100 μm; E and F = 50 μm; G (for G to J) = 25 μm.

Figure 6.

WUS1 fully complements PRS1 function. When expressed from the native PRS1 promoter, WUS1 (C5 WUS) fully complements all phenotypes (A, E, and I, respectively). l, Lateral sepals; ad, adaxial sepals; ab, abaxial sepals; arrows, lateral stipules. Bars in A = 3 cm; B (for B to D) = 100 μm; E (for E to G) = 50 μm.

Figure 7.

NS proteins accumulate in the L1 and L2 layers of the maize SAM. A, In situ hybridization analysis of a transverse section through the maize shoot apex reveals ns mRNA expression (purple) in two lateral foci in the L1 layer of the SAM. B, Immunohistolocalization analyses of slightly oblique, non-median section through the maize shoot apex reveals NS protein accumulation (purple) in two lateral foci, but in the L1-L2 layers. The image shown in A was generated by J. Nardmann and W. Werr and reproduced from Nardmann et al. (2004). Bar = 25 μm.

Figure 8.

Model for WOX3 accumulation and lateral non-cell autonomy in maize and Arabidopsis. A, Leaf development from the maize SAM. Polar auxin transport is required to initiate maize leaf development at the midrib region and recruit founder cells for the central domain (green). Accumulation of NS protein localizes to two lateral foci (red), although NS functions non-cell autonomously to recruit founder cells in the much larger margin domain shown in yellow (Scanlon, 2000). Loss of NS function generates mutant half leaves in which the entire margin domain is deleted (Scanlon et al., 1996). B, Sepal development in stage 3 floral meristem (FM) in Arabidopsis. The adaxial and abaxial sepals initiate first (green). PRS1 accumulates at two lateral foci (red) to initiate recruitment of lateral sepal founder. Genetic analyses (Matsumoto and Okada, 2001) reveal that PRS1 functions non-cell autonomously in a larger lateral domain (yellow) to effect recruitment of founder cells that give rice to the entire lateral sepals in addition to the margins of the adaxial and abaxial sepals.